Conformational Stabilities of the Structural Repeats of Erythroid Spectrin and Their Functional Implications

An, Xiuli; Guo, Xinhua; Zhang, Xihui; Baines, Anthony J.; Debnath, Gargi; Moyo, Damali; Salomao, Marcela A.; Bhasin, Nishant; Johnson, Colin G.; Discher, Dennis E.; Gratzer, Walter; Mohandas, Narla

doi:10.1074/jbc.m513725200

Cited by 64 publications

(82 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Binding of RESA to ␤1R16 spectrin structural repeat close to the interaction region between ␣1 and ␤1 spectrins significantly increases the stability of spectrin tetramers (24). In healthy RBCs, the spectrin network exists in a dynamic state of spectrin tetramers dissociating transiently into dimers, and shear deformation shifts the equilibrium of tetramer formation toward the dissociated dimer state (25). Thus, RESA stabilization of tetramers could modify spectrin network dynamic properties, thereby impairing membrane deformability.…”

Section: Discussionmentioning

confidence: 99%

Effect of plasmodial RESA protein on deformability of human red blood cells harboring Plasmodium falciparum

Mills¹,

Diez-Silva

Quinn

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

179

166

View full text Add to dashboard Cite

During intraerythrocytic development, Plasmodium falciparum exports proteins that interact with the host cell plasma membrane and subplasma membrane-associated spectrin network. Parasiteexported proteins modify mechanical properties of host RBCs, resulting in altered cell circulation. In this work, optical tweezers experiments of cell mechanical properties at normal physiological and febrile temperatures are coupled, for the first time, with targeted gene disruption techniques to measure the effect of a single parasite-exported protein on host RBC deformability. We investigate Pf155/Ring-infected erythrocyte surface antigen (RESA), a parasite protein transported to the host spectrin network, on deformability of ring-stage parasite-harboring human RBCs. Using a set of parental, gene-disrupted, and revertant isogenic clones, we found that RESA plays a major role in reducing deformability of host cells at the early ring stage of parasite development, but not at more advanced stage. We also show that the effect of RESA on deformability is more pronounced at febrile temperature, which ring-stage parasite-harboring RBCs can be exposed to during a malaria attack, than at normal body temperature. malaria ͉ erythrocyte ͉ membrane shear modulus ͉ spectrin ͉ cytoskeleton A fter Plasmodium falciparum merozoite invasion of a human RBC, the parasite differentiates and multiplies for 48 h, leading to rupture of the parasitized RBC (Pf-RBCs) and release of new merozoites in the blood circulation. Throughout this 48-h period, several parasite proteins are introduced into the RBC plasma membrane and submembranous protein skeleton, thereby modifying a range of structural and functional properties of the Pf-RBCs (1-4). The best documented changes occur as P. falciparum matures to the trophozoite (24-36 h) and schizont (36-48 h) stages, when Pf-RBCs display decreased membrane deformability (2-6), become spherical, and develop cytoadherence properties responsible for parasite sequestration in the postcapillary venules of different organs (7,8). In contrast, during early parasite development, ring-stage (0-24 h after invasion) Pf-RBCs preserve their biconcave shape, can circulate in peripheral blood (9), and thus are exposed to the spleen red pulp. Ring-stage Pf-RBCs may pass through this spleen compartment, be expelled from circulation, or return to the circulation once the parasite has been removed (10). Although the relative importance of these different processes and their underlying mechanisms are not fully understood, it is likely that altered deformability of ring-stage Pf-RBCs (11) plays a crucial role in determining Pf-RBCs spleen processing.Introduction of parasite components within the Pf-RBC membrane and cortical cytoskeleton begins soon after RBC invasion, as demonstrated for the well characterized parasite protein Pf155/Ring-infected erythrocyte surface antigen (RESA) (12). This protein is discharged by the invading merozoite and exported to the Pf-RBC membrane where, once phosphorylated, it interacts with the spectrin n...

show abstract

Section: Discussionmentioning

confidence: 99%

Effect of plasmodial RESA protein on deformability of human red blood cells harboring Plasmodium falciparum

Mills¹,

Diez-Silva

Quinn

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

179

166

View full text Add to dashboard Cite

show abstract

“…The design of all αII-and βII-spectrin single repeats and tandem-repeat fragments followed that for αI-and βI-spectrin fragments (20). The residue numbers of all sequences are given in Table 1.…”

Section: Design and Subcloning Of Recombinant Brain Spectrin Polypeptmentioning

confidence: 99%

“…Having previously found that the repeating units of αI-and βI-spectrin vary widely in their thermostabilities (20), we were prompted to examine those of αII-and βII-spectrins to investigate the basis of the differences in the gross characteristics of the intact proteins. We have accordingly determined the thermal unfolding properties of each repeat of αII-and βII-spectrin, as well as some constructs comprising selected tandem pairs of repeats, and some with mutations in the single α-helix uniting them.…”

mentioning

confidence: 99%

Thermal Stabilities of Brain Spectrin and the Constituent Repeats of Subunits

Zhang

Salomao

et al. 2006

Biochemistry

Self Cite

View full text Add to dashboard Cite

The different genes that encode mammalian spectrins give rise to proteins differing in their apparent stiffness. To explore this, we have compared the thermal stabilities of the structural repeats of brain spectrin subunits (αII-and βII) with those of erythrocyte spectrin (αI-and βI). The unfolding transition mid-points (T m ) of the 36 αII-and βII-spectrin repeats extend between 24 and 82°C, with an average higher by some 10°C than that of the αI-and βI-spectrin repeats. This difference is reflected in the T m -s of the intact brain and erythrocyte spectrins. Two of three tandem-repeat constructs from brain spectrin showed strong cooperative coupling, with elevation of the T m of the less stable partner corresponding to coupling free energies of about −4.4 and −3.5 kcal mol −1 . The third tandem-repeat construct, by contrast, showed negligible cooperativity. Tandem-repeat mutants, in which a part of the 'linker' helix that connects the two domains was replaced by a corresponding helical segment from erythroid spectrin, showed only minor perturbation of the thermal melting profiles, without breakdown of cooperativity. Thus the linker regions, which tolerate few point-mutations without loss of cooperative function, have evidently evolved to permit conformational coupling in specified regions. The greater structural stability of the repeats in αII-and βII-spectrin may account, at least in part, for the higher rigidity of brain compared to erythrocyte spectrin.Spectrin arose in evolution with the metazoa to meet the need for structures that strengthen cell adhesions and stabilize the plasma membrane against the forces of animal movement (1). The protein also plays a part in organizing plasma membrane signalling complexes (1, 2). Spectrin occurs as an (αβ) 2 tetramer, specialized for cross-linking actin filaments to membrane constituents. Both the α and β chains are largely made up of consecutive triplehelical repeating units of about 106 amino acids (3, 4); these act in ensemble as spacers between actin-binding domains in the β-subunits at opposite ends of the tetramers, and some contain binding sites for proteins such as ankyrin or for aminophospholipids (5, 6). *Author for correspondence: Xiuli An, Red Cell Physiology Laboratory, 310 E 67 th St, New York, NY10021, Tel: 212-570-3247; Fax: 212-570-3195. xan@nybloodcenter.org. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe number of spectrin genes increased during evolution with the advent of vertebrates.Invertebrates have one α-spectrin and, one β-spectrin with 16 complete triple-helical repeats and a β Heavy subunit with 30 complete triple helices. Vertebrates have four genes encoding 'conventional' β subunits (βI-IV) that have 16 complete triple helical modules, and one β Heavy subunit that has 30 triple helices (βV-spectrin) (1). Mammals have gained an additional α-spectrin by duplication of the pre-existing α-spectrin gene (7). There is now clear evidence of functional specialization of the two mammalian α-...

show abstract

“…1A. All recombinant proteins were expressed at 16°C, at which the yield was optimal, and allowed the isolation of soluble products in a state of high purity (11,26). The structure of the PfEMP3 protein is illustrated in Fig.…”

Section: Mapping the Pfemp3 Binding Site In Spectrin-mentioning

confidence: 99%

Plasmodium falciparum Erythrocyte Membrane Protein 3 (PfEMP3) Destabilizes Erythrocyte Membrane Skeleton

Pei

Guo

Coppel

et al. 2007

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) is a parasite-derived protein that appears on the cytoplasmic surface of the host cell membrane in the later stages of the parasite's development where it associates with membrane skeleton. We have recently demonstrated that a 60-residue fragment (FIa1, residues 38 -97) of PfEMP3 bound to spectrin. Here we show that this polypeptide binds specifically to a site near the C terminus of ␣-spectrin at the point that spectrin attaches to actin and protein 4.1R in forming the junctions of the membrane skeletal network. We further show that this polypeptide disrupts formation of the ternary spectrin-actin-4.1R complex in solution. Importantly, when incorporated into the cell, the PfEMP3 fragment causes extensive reduction in shear resistance of the cell. We conjecture that the loss of mechanical cohesion of the membrane may facilitate the exit of the mature merozoites from the cell.The intraerythrocytic form of Plasmodium falciparum, the agent of the most severe type of human malaria, exports many (according to a proteomic estimate, up to 400) proteins into the host cell in the course of its growth and development (1-3). Certain of these proteins are known to associate with the host cell membrane skeleton and thereby modify its structure and properties. Among the changes that have been reported are an altered morphology, an increased membrane rigidity, and an enhanced propensity to adhere to the vascular endothelium (4). Several such proteins that bind to the red cell membrane skeleton have so far been characterized. When the parasite first invades red cells, it deposits RESA (the ring parasite-infected erythrocyte surface antigen) at the membrane skeleton of the newly invaded cell where it binds to spectrin and protects against thermal damage (5-7). As the intracellular parasite further matures, it traffics further proteins to the skeleton. P. falciparum erythrocyte membrane protein 1, or PfEMP1, 2 is inserted into the red cell membrane and its extracellular domain adheres to receptors on endothelial cells (8). PfEMP1 clusters on the red cell surface at dimpled areas called knobs by binding of its intracellular domain to the parasite-encoded protein KAHRP (knob-associated histidine-rich protein) from which knobs are formed (9) and to spectrin and actin (10). KAHRP itself is stabilized at the membrane by interactions with the fourth repeat of spectrin ␣-chain (11). MESA (the mature parasite-infected erythrocyte surface antigen) binds to the 30-kDa domain of protein 4.1R and displaces the host protein p55 (12, 13). Its role in parasite biology is still uncertain, but interference with MESA binding is lethal to the parasite (14). The P. falciparum erythrocyte membrane protein 3 (PfEMP3), with which we are concerned here, is a large protein of 315 kDa that is expressed from the late ring stage onward and is exported via the Maurer's clefts, vesicular structures of parasite origin, into the cytoplasm of the red cell, where it associates with membrane skeleton. H...

show abstract

Conformational Stabilities of the Structural Repeats of Erythroid Spectrin and Their Functional Implications

Cited by 64 publications

References 33 publications

Effect of plasmodial RESA protein on deformability of human red blood cells harboring Plasmodium falciparum

Effect of plasmodial RESA protein on deformability of human red blood cells harboring Plasmodium falciparum

Thermal Stabilities of Brain Spectrin and the Constituent Repeats of Subunits

Plasmodium falciparum Erythrocyte Membrane Protein 3 (PfEMP3) Destabilizes Erythrocyte Membrane Skeleton

Contact Info

Product

Resources

About